The Triggering Receptors Expressed on Myeloid Cells (TREM) are expressed on a variety of innate immune cells including monocytes, macrophages, dendritic cells (DC), neutrophils, and osteoclasts. These receptors deliver signals to their host cells via association with the signaling chain, DAP12. DAP12 signaling is dependent on the presence of an immunoreceptor tyrosine-based activation motif (ITAM) within its cytoplasmic tail. Upon stimulation of a DAP12-coupled receptor, DAP12 is phosphorylated and recruits proteins critical to the propagation of downstream signals. Recent work has demonstrated that members of the TREM family, via DAP12, can deliver either activation or inhibitory signals to monocytes and macrophages. However, the biochemical nature of DAP12 signaling within myeloid cells is largely uncharacterized as is the overall immunological role of the TREM gene cluster. Thus, we have taken a bipartite approach to understand the immunobiology of the TREM cluster. Our second approach to understanding the role of TREM in regulation of innate immunity and cancer is dissection of the DAP12 signaling pathway in myeloid cells. Potential Environmental Impact on Tyrobp phenotypes. Over the years, we have been particularly interested in the apparent paradoxical activities of DAP-12-coupled receptors. As noted above, TREM-1 reportedly synergizes with TLR signals to promote inflammation in neutrophils, macrophages, and DC. In contrast, TREM-2, which also signals via DAP-12, has been shown to dampen inflammatory responses in DCs and macrophages. In addition, there are conflicting data regarding the role of DAP-12 in autoimmune diseases. Early reports suggested that Tyrobp-/- mice were resistant to experimental autoimmune encephalitis (EAE), whereas antibody to TREM-2 exacerbates the disease and Tyrobp-/- are reportedly more sensitive to immune-mediated diabetes. In a collaborative effort with the National Eye Institute, we tested the susceptibility of Tyrobp-/- mice to the induction of experimental autoimmune uveitis (EAU). qPCR demonstrated substantial expression of Tyrobp and Trem2 prior to immunization, and upregulation of multiple DAP-12-coupled receptors during peak EAU. Consistent with the anti-inflammatory activity of DAP-12 when coupled to TREM-2, Tyrobp-/- mice initially showed increased EAU disease scores relative to wild-type mice. To our surprise and dismay, re-derivation of these mice into a new, cleaner animal facility completely reversed this phenotype. Subsequent experiments showed no change in the EAU of wild-type mice, but Tyrobp-/- mice were now hyporesponsive to immunization-induced disease. These findings are consistent with modification of TREM-mediated phenotypes by underlying environmental factors. TREM-Like Transcript-4 as a Potential New Player in Human Disease. Previous work on Treml4 has focused on the murine receptor. The syntenic human gene contains an apparent open reading frame predicted to encode a TREM-like protein with a non-canonical leader sequence, conserved V-set Ig domain, short stalk and a hydrophobic domain that is truncated just distal to the lysine residue that would be predicted to facilitate interaction with DAP12. Few ESTs exist within public databases, giving little clue as to the function of TREML4 in humans. We collaborated with Shurjo Sen, of the national Human Genome Research Institute of NIH, who initiated an RNA Seq case-control study of coronary artery calcification (CAC) using a large network of characterized donors known as ClinSeq. This unbiased approach identified multiple genes with apparent association with CAC. The top four immune genes were TREML4, and three KIR loci associated within B-type KIR haplotypes. Working closely with NHGRI, we characterized two SNPs that are associated with mRNA expression for TREML4 in humans, rs2803495 and rs2803496. Using these SNPs we defined three apparent haplotypes, those with a rare variant at rs2803495 but common at rs2803496, those common at rs2803495 but rare at rs2803496, and the majority of donors with the common variants at both locations. We found substantial TREML4 mRNA only in donors who were carriers of at least one copy of a rare allele, either rs2803495 or rs2803496; thus we term these alleles permissive. Similar to mouse, we found that TREML4 mRNA could be detected in purified monocytes and neutrophils from donors with permissive alleles, the latter having approximately 100-fold higher expression. Molecular analysis of the TREML4 mRNA confirmed a mRNA of 1.8 kb with an intact open reading frame. Transfection experiments showed that when armed with an effective leader sequence, the transmembrane domain was capable of anchoring TREML4 to the cell surface. However, the native TREML4 leader is incapable of effectively targeting the protein to the cell surface, suggesting that TREML4 may regulate neutrophil function as an intracellular or secreted protein or via RNA-mediated mechanisms. Future plans for Treml4 are collaborative studies with the Biesecker lab at NHGRI, the laboratory that initiated the work on coronary artery calcification, and with Dr. Frank Kolodgie at CV Path Institute, a nonprofit pathology based organization that specializes in the pathological analysis of cardiovascular disease and are not detailed here.